Hypochlorite is defined herein to include any bleach such as Clorox.RTM. which generate hypochlorite anion, OCl.sup.--, upon addition to water and any hypochlorite generated by mixing chlorine in water as shown below: ##STR1## In alkaline solution: EQU Cl.sub.2 +2OH.sup.-- .fwdarw.Cl.sup.-- +OCl.sup.-- +H.sub.2 O
Many enzymes have been found to be inactivated in the presence of chlorine. For example, a paper by Waku et al, CA 78(17):107533p, discloses the inactivation of digestive enzymes trypsin, chymotrypsin, pepsin, pancreatic amylase and pancreatic RNase in concentrations as small as 0.2 ppm of free chlorine.
Enzymes such as proteases are widely used in industrial settings. Inactivation at this low concentration of chlorine could then occur if chlorinated city water is used to clean industrial production equipment. In the dairy or brewing industry, city water, sometimes with the addition of Clorox.RTM. bleach, is used in cleaning equipment. Even a low concentration of hypochlorite left in such equipment could inactivate the proteases used to prepare cheese, yogurt or other fermented milk products.
A specific example of enzyme inactivation in the presence of hypochlorite is found in alkaline proteases which are well known detergent additives. These proteases are sensitive to oxidation. Most are inactivated by very low concentrations of bleach and some are even inactivated by so called "color safe" bleaches, i.e. perborate and peroxy bleaches.
Suggestions have been made to improve the properties of alkaline proteases by site directed mutagenesis. In European Patent Application 0130756 published Jan. 9, 1985, a method for preparing an improved carbonyl hydrolase by site directed mutagenesis was disclosed. The application suggests that oxidative stability, pH activity profiles, K.sub.m, k.sub.cat, k.sub.cat /K.sub.m ratios and substrate specificity can be improved by the substitution, deletion or insertion of at least one amino acid at a predetermined site in the hydrolase. Specifically it was suggested that enhanced oxidative stability can be effected by deleting one or more methionine, tryptophan, cysteine or lysine residues and, optionally, substituting another amino acid residue not one of methionine, tryptophan, cysteine or lysine, preferably alanine but alternatively neutral residues.
However, alkaline proteases contain from about 250 to about 290 amino acids and may contain 15 to 18 sites which fit the description above. It would require an inordinate amount of experimentation to perform site directed mutagenesis at each of those sites and to express and test each substitution for improved oxidative stability.
The method of this invention provides a means to identify a site of oxidative cleavage and has been used to identify a specific residue which can be altered to confer the desired stability. The method may be applied to any enzyme which is cleaved in the presence of chlorine and is particularly applicable to alkaline proteases.